Iron plays a critical role in both the healthy and diseased retina. The long term goals of the proposed studies are to understand regulation of retinal iron flux, determine why iron accumulates in retinal disease, and discover how to protect against retina iron toxicity. Iron is necessary in the retina for oxidative phosphorylation, membrane biogenesis and retinol isomerization, but becomes a central producer of oxidative stress when improperly regulated. Iron toxicity is evident in retinal disease as follows: 1) Iron causes rapid retinal degeneration following entry into the eye carried by an intraocular foreign body. 2) Human AMD retinas have more iron than age-matched controls, suggesting that iron overload may play a role in AMD pathogenesis. 3) Consistent with this hypothesis, in the inherited disease aceruloplasminemia, loss of the ferroxidase ceruloplasmin (Cp) results in retinal iron accumulation and early onset macular degeneration. 4) Mice with knockout for Cp and its homolog hephaestin (Heph) have an age-dependent retinal iron overload and degeneration sharing features of AMD, including complement activation and subretinal neovascularization. The latter two points indicate that Cp and Heph are important for retinal health. Evidence from other organs suggests that Cp or Heph can cooperate with the plasma membrane iron transporter ferroportin (Fpn) to export iron from cells. Progress from our prior funding period indicates that Heph plays a cell-autonomous role in RPE iron export, but that it also has a critical function in inner retinal iron transport. One goal of the current proposal is t discover the role of Muller cell Heph in retinal iron regulation. Another goal is to test the hypothesis that a third, recently identified ferroxidase, amyloid precursor protein (APP), mediates iron import into the retina. The balance of retinal iron importers and exporters is controlled by iron regulatory proteins (IRPs). These can be dysregulated in disease by hypoxia or oxidative stress. We will assess the role of IRPs in retinal iron regulation in healthy and diseased retinas. The synergistic proposed studies using primary cell culture, systemic and cell-type specific conditional knockouts and post mortem human tissues will increase our understanding of the effects of ferroxidases and IRPs on retinal iron in health and disease.